Retroviral vector particles are useful agents for transducing polynucleotides into cells, such as eukaryotic cells.
Thus, retroviral vector particles have been used for introducing polynucleotides into cells for gene therapy purposes. In one approach, cells are obtained from a patient, and retroviral vector particles are used to introduce a desired polynucleotide into the cells, and such modified or engineered cells are returned to the patient for a therapeutic purpose. In another approach, retroviral vector particles may be administered to the patient in vivo, whereby the retroviral vector particles transduce cells of the patient in vivo.
In many gene therapy protocols, it would be desirable to target retroviral vector particle infection to a specific population of cells either in vivo or in vitro. In such circumstances, the broad host range of typical retroviruses present a significant problem. A key determinant of viral host range is the “envelope” or “env” protein (encoded by the env gene) which is involved in binding to receptors on the surface of susceptible cells. Where it is possible to purify the desired target cells, either before or after transduction, such purification necessitates undesirable manipulations of the cells and may be problematic in situations in which the preferred target cells either are difficult to purify or are present at low or variable frequencies in mixed cell populations. Thus, it would be advantageous to have retroviral vector particles which could infect particular types of mammalian cells.
Retroviral vectors have been made which have modified envelopes; however, such vectors in general are less infective than wild-type retroviral vectors or retroviral vectors including foreign genes, but which have unmodified envelopes.
Attempts to insert large, complex, or bulky polypeptides such as single chain antibodies, polypeptide ligands, or complement regulatory proteins have in the past been hampered by poor expression, incorporation, folding, and/or presentation of the chimeric env proteins. The present invention provides “modified env proteins” that permit the incorporation, expression and assembly of large polypeptides within the basic framework (i.e. N-terminal signal peptide, N-terminus, surface (SU) C-terminus and membrane spanning transmembrane (TM) domains) of the env protein of a virus, for example a retrovirus. These modified env proteins are devoid of much of the receptor binding domains. Hereinafter such proteins will be referred to as “escort proteins”. Escort protein necessarily requires co-expression with a wild type env to gain infectivity. The “escort protein” provides the gain of function; i.e. targeting motif that directs or escorts the virus to the specific target cell or target ligand, such as IgG or exposed collagen or ECM.
A definition of the following terms is provided for the avoidance of doubt.
A “retroviral vector particle” is an infectious virion derived from a retrovirus.
A “retroviral vector plasmid vector” is a non-infectious plasmid comprising retroviral DNA, wherein said plasmid is capable of use as a vector for transfection of a target cell “Retroviral DNA” is DNA transcribed from retroviral RNA by reverse transcriptase.